Real-Time DNA Sequencing Using Detection of Pyrophosphate Release

Abstract An approach for real-time DNA sequencing without the need for electrophoresis has been developed. The approach relies on the detection of DNA polymerase activity by an enzymatic luminometric inorganic pyrophosphate (PP i ) detection assay (ELIDA) (Nyren, P. (1987) Anal. Biochem. 167, 235–238). The PP i formed in the DNA polymerase reaction is converted to ATP by ATP sulfurylase and the ATP production is continuously monitored by the firefly luciferase. In the sequencing procedure, immobilized single-stranded template was used in a repeated cycle of deoxynucleotide extension. Real-time signals in the ELIDA, proportional to the amount of incorporated nucleotide, were observed when complementary bases were incorporated. An increased signal-to-noise ratio was obtained by substitution of deoxyadenosine α-thiotriphosphate (dATPαS) for the natural deoxyadenosine triphosphate, dATPαS is efficiently used by the DNA polymerase, but is not recognized by the luciferase. As a model, 15 bases of a single-stranded PCR product were sequenced. The possibility for parallel processing of many samples in an automated manner is discussed.